The Role of ATP on Reactive Oxygen Species Generation and Cellular Proliferation in BV-2 Cells / 대한소아신경학회지

PURPOSE: Adenosine triphosphate (ATP) evokes several cellular responses in microglia including propagation. However, the role of the purinoceptor on ROS generation in microglia is unclear. In order to determine the action of the purinoceptor in microglia, the effects of ATP on ROS generation and cellular proliferation in BV-2 murine microglial cells were evaluated. An additional aim of this study was to investigate signal transduction pathways using several inhibitors. METHODS: The [Ca2+] was measured using Ca2+ sensitive indicator, Fura-2/AM. ROS production was observed by fluorescence-confocal microscope and cell proliferation was evaluated by counting cell number. RESULTS: ATP increased the intracellular calcium levels ([Ca2+]i) in BV-2 cells in a dose-dependent manner. This increase was attenuated by pretreatment with a calcium chelator (EGTA) and a phospholipase C (PLC) inhibitor (U-73122) while the protein tyrosine kinase (PTK) inhibitor (genistein) had no inhibitory effects. To identify the effects of the nucleotides, ROS generation was observed in the nucleotide-stimulated BV-2 cells. The treatment with 100 M ATP induced ROS generation, but 100 M adenosine and 100 M UTP did not. To investigate the signal transduction pathway in ATP-induced ROS generation, several inhibitors were pretreated before adding ATP. ATP- induced ROS production was blocked by pretreatment with either 0.5 mM EGTA or 10 M U73122 while 40 M genistein had an inhibitory effect on ATP action. Correspondingly, 40 M KN62 (CaM kinase II inhibitor), 1 M sphingosine (protein kinase C inhibitor), 1 nM DPI (NADPH oxidase inhibitor) and 50 M mepacrine (phospholipase A2 inhibitor) could suppress ATP-induced ROS generation. The effects of ATP on cell proliferation was observed 3 days after ATP treatment and its peak velocity after 4 days. NF-kB activation was observed after the cells were incubated with 0.1 mM ATP. The maximal level of NF-kB activation was obtained with 0.3 mM ATP while higher concentrations were less effective. CONCLUSION: Overall, we conclude that ATP in BV-2 cells induces ROS generation and cell propagation. The signal transduction pathways including calcium, CaM kinase II, PLC, protein kinase C, phospholipase A2 and NADPH oxidase are involved in ATP-induced ROS generation.

Varying Effects of Intravenous Immunoglobulin on Mononuclear Cell Proliferation In Vitro

Intravenous immunoglobulin (IVIG) is being increasingly used to treat numerous immune-mediated diseases. However, there is a paucity of knowledge on the specific mode of action of IVIG in vivo. In this study, the in vitro effects of IVIG on peripheral blood mononuclear cell (PBMC) proliferation using phytohemagglutinin (PHA), anti-CD3 monoclonal antibody (MAb), phorbol myristate acetate (PMA), or purified protein derivatives (PPD) have been analyzed. The PBMCs were obtained from more than 10 individual donors. In all cases, IVIG almost completely inhibited PBMC proliferation at concentration above 20 mg/mL except when used in conjunction with PMA. PHA-induced proliferation of PBMCs at concentrations ranging from 1 to 15 mg/mL did not show significant differences. Anti-CD3 MAb-induced proliferation showed dose-dependent inhibition at concentrations ranging from 1 to 10 mg/mL. Interestingly, PMA-induced proliferation of PBMCs showed a dose-dependent increase at the same concentration range. PPD-induced proliferation of PBMC at concentrations ranging from 1 to 10 mg/mL did not show any statistically significant differences. These results suggest that high dose IVIG may be necessary to immune modulation in vivo and IVIG has various effects on PBMCs proliferation in limited concentration in vitro.

Effects of Vigabatrin on Pilocarpine-Induced Seizures in Immature Rats

PURPOSE: Vigabatrin is a widely used antiepileptic drug that greatly increases whole brain gamma- aminobutyric acid(GABA). But little is known about the anticonvulsant effect of vigabatrin on pilocarpine-induced seizures in the immature rats. This study was conducted to determine the effects of vigabatrin on pilocarpine-induced seizures in the immature rats. METHODS: Six to eight day old Sprague-Dawley rats were classified into control(n=5) and vigabatrin-treated(n=5) groups that were pretreated with 30mg/kg of vigabatrin. Animals received vigabatrin or saline, intraperitonealy, for 6 days, once a day. And on the 5th day, right and left cortical electrodes were placed in 10-14 day old animals using stereotaxic instrument. The following day 2.5-hour EEG recordings were obtained to monitor the latency to first electrographic seizures and to first status epilepticus induced by intraperitoneal injection of pilocarpine(200mg/kg). Data were analyzed using the log-rank test. RESULTS: Electrographic seizures and status epilepticus were seen in 80% of vigabatrin-treated group, and in 100% of control group rats. And the latency to first seizure was 8.8+/-2.0 minutes in control group and 20.5+/-5.2 minutes in vigabatrin-treated animals(P<0.02), and to status epilepticus was 12.2+/-1.2 minutes in control group and 29.3+/-6.3 minutes in vigabatrin-treated group(P<0.03). CONCLUSION: It was confirmed that 30mg/kg of vigabatrin administration for 6 days did not affect the body weight gain and behavior of immature rats and had an anticonvulsant effect. These findings might demonstrate that the prolonged latency to seizure, and to status epilepticus, was a time to reduce GABA that was elevated in the brain by vigabatrin administration below the seizure threshold, by pilocarpine.

A Case of Common Variable Immunodeficiency with Autoimmune Hemolytic Anemia

Common variable immunodeficiency (CVID) is a heterogeneous collection of disorders with hypogammaglobulinemia with recurrent bacterial infections and high incidence of autoimmune disorders as its hallmark. We report a 7-year-old girl suffering from CVID with Coombs' test positive hemolytic anemia. She had been relatively well until 23-months old when she was admitted to Taejon St. Mary's Hospital with pneumonia 5 years ago. Afterwards, she had suffered from recurrent otitis media, paranasal sinusitis, bronchitis and pneumonia, experiencing 13 admissions. She was diagnosed as autoimmune hemolytic anemia at 4-years old and had been treated with prednisolone. Laboratory finidings showed hypogammaglobulinemia(gamma-globulin in immunoelectrophoresis 0.04g/dL, IgG 170mg/dL, IgA 31mg/dL, IgM 27.5mg/dL) which was previously within normal limits checked at the age of 3- and 5-years old. Isohemmagglutinins (Anti-A,-B IgM and IgG) and anti-measles IgG, anti-mumps IgG, anti-rubella IgG and anti-HBs antibody along with PPD skin test were all negative. Peripheral lymphocyte subsets revealed as follows : pan T cells (CD3+) 48.6% (normal values : 60-85%), pan B cells (CD19+) 36.7% (8-20%), CD4+ T cells 24.4% (28+/-8%), CD8+ T cells 15.3% (5+/-10%), and CD4/CD8 ratio of 1.6 (0.6-2.8). Proliferations of peripheral blood mononuclear cells induced by various T cell stimulants were all markedly depressed. Chronic paranasal sinusitis and lung parenchymal damages were revealed on computerized tomography and lung scan, and a monthly intravenous immunoglobulin therapy was started.

A Case of Glucose-6-phosphate dehydrogenase Riley Causing Hemolytic Anemia / 대한혈액학회지

A glucose-6-phoshate dehydrogenase variant called G6PD Riley was detected in an Korean boy with nonspherocytic hemolytic anemia. Using polymerase chain reaction based single-strand conformation polymorphism (PCR-SSCP) followed by DNA sequence analysis, we found mutation T to C at nucleotide 1139 in exon 10, resulting in a substitution of 380th amino acid isoleucine to threonine. The patient's mother was confirmed to be a heterozygote.

A Case of Hemimegalencephaly with Focal Seizure / 대한소아신경학회지

Hemimegalencephaly is a rare brain malformation characterized by congenital hypertrophy of one cerebral hemisphere, ipsilateral ventriculomegaly, hemiparesis, intractable epilepsy, and mental retardation, which often results in early death. We reported a case of hemimegalencephaly in a 1-month-old male with the chief complaint of intractable focal seizure.

A Case of Acute Drug-Induced Agranulocytosis Treated with Recombinant Granulocyte Colony-Stimulating Factor(G-CSF)

Drug-induced agranulocytosis is a potentially lethal disorder characterized by selective neutropenia. G-CSF has been utilized for its treatment. We report a case of acute agranulocytosis probably associated with injection of sulpyrine(dipyrone). A three-year old girl was admitted to Taejon St. Mary's Hospital following five days of fever and two days of chills and prostration. During this period, she had been treated at local clinics with oral acetaminophen, ibuprofen, and miokamycin. Two days before admission, she was administered an intramuscular injection of dipyrone as antipyretics. She had a past history of previous sensitization of dipyrone. CBC revealed profound netropenia(total WBC 900/mm3, with 1% neutrophils, 88% lymphocytes, 10% atypical lymphocytes, 1% monocytes), but normal RBC and platelet count. Bone marrow examination showed hypocellularity(20%), decreased myeloid precusors, and M:E ratio of 1 : 2.5. The girl received subcutaneous G-CSF once daily for 3 days. G-CSF therapy resulted in a steep increase of neutrophil count, which was faster than the spontaneous recovery reported in the literature. G-CSF may be considered useful in the management of drug-induced agranulocytosis.

A Case of Acute Drug-Induced Agranulocytosis Treated with Recombinant Granulocyte Colony-Stimulating Factor(G-CSF)

Drug-induced agranulocytosis is a potentially lethal disorder characterized by selective neutropenia. G-CSF has been utilized for its treatment. We report a case of acute agranulocytosis probably associated with injection of sulpyrine(dipyrone). A three-year old girl was admitted to Taejon St. Mary's Hospital following five days of fever and two days of chills and prostration. During this period, she had been treated at local clinics with oral acetaminophen, ibuprofen, and miokamycin. Two days before admission, she was administered an intramuscular injection of dipyrone as antipyretics. She had a past history of previous sensitization of dipyrone. CBC revealed profound netropenia(total WBC 900/mm3, with 1% neutrophils, 88% lymphocytes, 10% atypical lymphocytes, 1% monocytes), but normal RBC and platelet count. Bone marrow examination showed hypocellularity(20%), decreased myeloid precusors, and M:E ratio of 1 : 2.5. The girl received subcutaneous G-CSF once daily for 3 days. G-CSF therapy resulted in a steep increase of neutrophil count, which was faster than the spontaneous recovery reported in the literature. G-CSF may be considered useful in the management of drug-induced agranulocytosis.

A Case of Glycogen Storage Disease Type IIa

We experienced a case of glycogen storage disease(type Iia) in a 11 months old girl who was admitted to Pediatric service of Kangnam St. Mary's hospital for work-up of flaccidity and developmental delay. The baby was relatively well until 3 months ol age when she began to have poor sucking and swallowing, and also her crying was weak. The patient has been markedly behind in all her developmental milestones and revealed hypotonia uhich was apparent on ventral and vertical suspensions. The chest X-ray film showed cardiomegaly, and echocardiography was done twice to get a diagnosis of hypertrophic cardiomyopathy. ECG showed biventricular hypertrophy. The brain CT showed no abnormality. Needle EMG showed fibrillation and positive sharp waves typical of a myopathy. Total CPK was 349 IU/L with an increase in fraction of MM band. Light and electronmicroscopic findings of muscle biopsy were compatible with Pompe's disease of infantile type.